STRUCTURAL PROPERTIES OF PHYCOERYTHRIN FROM DULSE PALMARIA PALMATA

We found that the red alga dulse (Palmaria palmata) contains a lot of proteins, which is mainly composed of phycoerythrin (PE) and the protein hydrolysates showed high angiotensin I converting enzyme (ACE) inhibitory activities. Therefore, we investigated the structure of dulse PE to discuss its structure-function relationship. We prepared the chloroplast DNA and analyzed the nucleotide sequences encoding PE by cDNA cloning method. It was clarified that dulse PE has alpha- and beta-subunits and they are composed by 164 amino acids (MW: 17,638) and 177 amino acids (MW: 18,407), respectively. The dulse PE contained conserved cysteine residues for chromophore attachment site. On the alignment of amino acid sequences of dulse PE with those of other red algal PE, the sequence identities were very high (81-92%). In addition, we purified and crystallized the dulse PE, and its crystal structure was determined at 2.09 angstrom resolution by molecular replacement method. The revealed 3D structure of dulse PE which forms an (alpha beta)(6) hexamer was similar to other red algal PEs. Conversely, it was clarified that the dulse PE proteins are rich in hydrophobic amino acid residues (51.0%), especially aromatic amino acid and proline residues. The data imply that the high ACE inhibitory activity of dulse protein hydrolysates would be caused by the specific amino acid composition and sequence of dulse PE

Flexible NAD(+)Binding in Deoxyhypusine Synthase Reflects the Dynamic Hypusine Modification of Translation Factor IF5A

The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD(+)and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure fromPyrococcus horikoshii OT3(PhoDHS) at 2.2 angstrom resolution. The structure reveals two states in a single functional unit (tetramer): two NAD(+)-bound monomers with the NAD(+)and spermidine binding sites observed in multi-conformations (closed and open), and two NAD(+)-free monomers. The dynamic loop region V288-P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD(+)is absent. Combined with NAD(+)binding analysis, it is clear thatPhoDHS can exist in three states: apo,PhoDHS-2 equiv NAD(+), andPhoDHS-4 equiv NAD(+), which are affected by the NAD(+)concentration. Our results demonstrate the dynamic structure ofPhoDHS at the NAD(+)and spermidine binding site, with conformational changes that may be the response to the local NAD(+)concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A

Rapid and Simple Detection of <i>Burkholderia gladioli</i> in Food Matrices Using RPA-CRISPR/Cas12a Method

Pathogenic variants of Burkholderia gladioli pose a serious threat to human health and food safety, but there is a lack of rapid and sensitive field detection methods for Burkholderia gladioli. In this study, the CRISPR/Cas12a system combined with recombinant enzyme polymerase amplification (RPA) was used to detect Burkholderia gladioli in food. The optimized RPA-CRISPR/Cas12a assay was able to specifically and stably detect Burkholderia gladioli at a constant 37 °C without the assistance of large equipment. The detection limit of the method was evaluated at two aspects, the genomic DNA (gDNA) level and bacterial quantity, of which there were 10−3 ng/μL and 101 CFU/mL, respectively. Three kinds of real food samples were tested. The detection limit for rice noodles, fresh white noodles, and glutinous rice flour samples was 101 CFU/mL, 102 CFU/mL, and 102 CFU/mL, respectively, without any enrichment steps. The whole detection process, including sample pretreatment and DNA extraction, did not exceed one hour. Compared with the qPCR method, the established RPA-CRISPR /Cas12a method was simpler and even more sensitive. Using this method, a visual detection of Burkholderia gladioli that is suitable for field detection can be achieved quickly and easily